Hereditary deficiency of galactose-1-phosphate uridylyltransferase (GALT, E.C. 2.7.7.12) activity can lead to a potentially lethal disease called Classic Galactosemia (OMIM 230400). Despite the life-saving consequences of newborn screening, early diagnosis, and a galactose-restricted diet, many patients with Classic Galactosemia suffer later in life from complications including growth, neuropsychological, and speech delays as well as premature ovarian insufficiency (POI). The pathogenic mechanisms of the acute lethality and the long-term complications associated with this disorder remain largely unknown, thus further hamper the development of more effective therapies. Yet, several lines of evidence suggested that elevated level of galactose-1-phosphate (gal-1P), product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in Classic Galactosemia. We, therefore, hypothesize that prevention of gal-1P production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, we, in collaboration with th High-throughput Screening (HTS) Facility at the NIH Chemical Genomics Center (NCGC), had launched a quantitative high-throughput screening (qHTS) campaign to identify small molecule inhibitors of the human GALK. To date, we have identified 149 small molecule compounds that, at micromolar (M) concentration, inhibit 80% or more of control GALK activity in vitro. Through biochemical, structure- and cell-based characterization of these compounds, we have identified a few promising chemotypes that are non- toxic and effective in lowering gal-1P accumulation in human GALT-deficient fibroblasts. The translation of these promising small molecule GALK inhibitors into safe and useful therapeutics, however, requires further chemical modifications and in vivo studies in mammalian animal models. In this application, we propose to set up an innovative, cost-effective, iterative and parallel optimization process to (1) Improve the biologicl activities and drug-like properties of the validated GALK inhibitors against GALK in vitro and in cell-based assays; (2) perform proof-of-concept (POC) studies by evaluating the efficacy of the optimized inhibitors in reducing gal-1P production galactose-challenged GalT-knockout mice. The completion of the proposed project will identify in vivo chemical probes for a novel target, GALK, which has significant implications ranging from the understanding of galactose metabolism in human organ development/ functions to the development of an improved therapy to address the unmet medical needs of the debilitating orphan disease, Classic Galactosemia. PUBLIC HEALTH RELEVANCE: Classic Galactosemia is a rare metabolic disorder included in all newborn screening programs in the USA. A galactose-restricted diet, which is currently the mainstay of treatment, can prevent neonatal lethality of the affected patients, but has little effet on long-term complications such as premature ovarian insufficiency and speech delay. The long-term goal of this project is to translate the validated small molecule inhibitors of human galactokinase (GALK) into novel, safe and more effective therapeutics for this debilitating disorder.